The transforming growth factor beta (TGF-.beta.) superfamily is a group of cytokines that regulate many aspects of cellular function. The structural prototype for the gene superfamily is TGF-.beta.. TGF-.beta. is produced as a precursor and the precursor structure is shared by most of the members of the TGF-.beta. superfamily. The superfamily includes the TGF-.beta. family, the inhibin family, the DPP/VGl family and the Mullerian Inhibiting Substance Family.
The TGF-.beta. family includes five members, termed TGF-.beta.1 through TGF-.beta.5, all of which form homodimers of about 25 kd (reviewed in Massague, Annu. Review Cell Biol. 6:597, 1990). The family also includes TGF-.beta.1.2 which is a heterodimer containing a .beta.1 and a .beta.2 subunit linked by disulfide bonds. The five TGF-.beta. genes are highly conserved over great evolutionary distances. The mature processed cytokines produced from the members of the gene family show almost 100% amino acid identity between species, and the five peptides as a group show about 60-80% identity.
All forms of TGF-.beta. have been found to reversibly inhibit growth activity in normal, epithelial, endothelial, fibroblast, neuronal, lymphoid, and hematopoietic cell types (For review see Massague, 1990, Annu. Review Cell Biol. 6:597). In tissue culture, TGF-.beta. has been shown to inhibit cell growth by blocking both cdk-4/cyclinD activation and cdk2/cyclinE activity, events required for G1 to S phase transition (M. E. Ewen, H. K. Sluss, L. L. Whitehouse, D. M. Livingston, Cell 74, 1009 (1993) and A. Koff, M. Ohtsuki, K. Polyak, J. M. Roberts, J. Massague, Science 260, 536 (1993)). The antiproliferative action of TGF-.beta. has also been demonstrated in vivo (Silberstein and Daniel, 1987, Science 237:291-93; and Russell et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5126-30).
TGF-.beta. can also stimulate cell proliferation although the effect may be secondary to other cellular responses. For example, TGF-.beta.1 has been shown to promote the anchorage independent growth of normal rat kidney fibroblasts (Roberts et al., PNAS U.S.A. 78:5339, 1981; Roberts et al, Nature 295:417, 1982 and Twardzik et al., 1985, J. Cell. Biochem. 28:289, 1985) and it induces colony formation of AKR-2B fibroblasts (Tucker et al. Science 226:705, 1984). The inhibitory/stimulatory actions of TGF-.beta. may depend on the cell type and the physiological state of the cells.
TGF-.beta. is involved in mediation of cell adhesion. TGF-.beta. action on normal mesenchymal, epithelial and lymphoid cells, and some tumor cell lines generally results in the up-regulation of cell adhesion. This up-regulation is mediated by enhanced synthesis and deposition of extracellular matrix components, decreased pericellular proteolysis, and modification of cell adhesion receptors (Massague, 1990).
Cellular differentiation processes of many cell lineages can be positively or negatively effected by TGF-.beta.. TGF-.beta. has been shown to exert positive effects on chondrocyte and osteogenic cell types (Massague, 1990).
The biological actions of TGF-.beta. described above suggest a broad role for TGF-.beta. in the physiologic setting. The ability of TGF-.beta. to modulate DNA replication, cell differentiation, cell adhesion and extracellular matrix layout indicate a role for TGF-.beta. in the generation and modification of signals that guide morphogenic events of embryogenesis. The activity of TGF-.beta. as a promoter of extracellular matrix formation and a regulator of cell migration and development, is a major influence in inflammation and tissue repair processes. In fact, the administration of TGF-.beta.1 into wound chambers or to incisional wounds has been shown to accelerate the wound healing response in general (Sporn et al., 1983, Science, 219:1329-31; Roberts et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:4167-71; and Mustoe et al., 1987, Science 237:1333-36).
The TGF-.beta. family of cytokines display an immunosuppressive activity in vitro and in vivo which is in part attributed to the antiproliferative action of TGF-.beta. on lymphocytes (Kehrl et al 1986, J. Immunol. 137:3855), T-lymphocytes (Kehrl et al 1986, J. Exp. Med. 163:1037) and thymocytes (Ristow 1986, PNAS U.S.A. 83:5531). An excess of TGF-.beta. activity in tissues may lead to an unbalanced deposition of extracellular matrix which may contribute to fibrosis, while a lack of TGF-.beta. growth inhibitory activity may lead to oncogenic transformation.
Dis-regulation of TGF-.beta. action has been implicated in the pathological processes of many diseases. TGF-.beta. has been reported to be a pathogenic mediator in HIV infections and its associated diseases (Lotz, M. and Seth, P. Annals of the N.Y. Acad. of Sciences 685:501, 1993).
TGF-.beta. has also been implicated in the development of fibrosis including pulmonary fibrosis (Baecher-Allan and Barth, Regional Immunology 5(3-4):207, 1993), and fibrosis associated with chronic liver disease (Burt, A. D., J. of Pathology 170:105, 1993), hepatic veno-occlusive and idiopathic interstitial pneumonitis, which are major causes of morbidity and mortality after bone marrow transplantation (Anscher M. S. et al. N.Engl.J.Med. 328:1592, 1993), kidney disease (Border et al., Nature 369:360:361, 1992), and radiotherapy or radiation accidents (Martin M. et al Radiation Research 134:63, 1993). TGF-.beta.2 has been found to be elevated in the eye of humans with proliferative vitreoretinopathy and in the skin in systemic sclerosis (Connor et al. J. Clin Invest. 83.:1661, 1989 and Kulozik et al., J. Clin. Invest. 86:917, 1990).
The use of TGF-.beta. and TGF-.beta. antagonists to modulate blood pressure is disclosed in PCT/US91/0449 published on Dec. 26, 1991. Recombinant TGF-.beta. obtained from Chinese Hamster Ovary cells was reported to induce rapid, significant, and sustained decreases in arterial blood pressure of cynomolgus monkeys receiving daily injections of the recombinant molecule.
The DPP/VGl family includes the bone marrow morphogenetic proteins (BMPs), termed BMP1 through BMP7, DPP and Vgl. The BMPs are osteoinductive agents present in adult bone. They are potent initiators of new bone formation and they appear to act on mesenchymal progenitor cells, directing their differentiation into both cartilage and bone-forming cells. It has also been suggested that they play an important role during embryonic skeleton formation (See Review by Rosen and Thies, in Trends in Genetics, 8(3)97, 1992 and references therein). Decapentaplegic (DPP) plays a fundamental role in dorsoventral body patterning and in imaginal disk formation in Drosophila (Padgett et al., 1987, Nature, 325:81-84). Vgl is involved in embryonic development in Xenopus laevis.
The inhibin family includes the activins and inhibins. The activins are involved in the regulation of numerous biological processes. For example, they are involved in the proliferation of many tumor cell lines, the control of secretion and expression of the anterior pituitary hormones (EG, FSH, GH, and ACTH), neuron survival, hypothalamic oxytocin secretion, erythropoiesis, placental and gonadal steroidogenesis early embryonic development and the like. Inhibin molecules also help to regulate erythropoiesis and modulate the release of FSH (Mason et al., 1985, Nature 318:659-663 and Ling et al., Nature 321:779, 1986).
The Mullerian Inhibiting Substance family includes Mullerian Inhibiting Substance (MIS) which is an important morphogenetic signal in developing reproductive systems of mammalian embryos (Cate et al, Cell 45:685-698).
The members of the TGF-.beta. superfamily may mediate signal transduction through families of related receptors. The cell surface receptors for TGF-.beta. and activin are composed of type I and type II receptor chains, both of which contain a cysteine rich extracellular domain and a cytosolic serine/threonine protein kinase domain. The type I and type II receptors form heterodimers which appears to be necessary for cytokine-dependent intracellular signalling (Massague, Cell, 69:1067, 1992 and references therein, and Lin et al., Cell 68:775, 1992).
TGF-.beta. binding proteins have been identified which do not function as signalling receptors. The proteoglycan betaglycan is the most widely distributed TGF-.beta. binding protein after receptors I and II. Betaglycan is a membrane anchored proteoglycan that binds TGF-.beta. via the core protein and it has been suggested that betaglycan acts as a receptor accessory molecule in the TGF-.beta. systems (Massague, Cell, 69:1067, 1992 and references therein).
A number of other extracellular matrix proteins have been shown to bind TGF-.beta., including decorin, biglycan, thrombospondin and the serum glycoprotein .alpha.2-macroglobulin (Y. Yamaguchi, D. M. Mann, E. Ruoslahti, Nature 346, 281 (1990); S. Scholtz-Cherry J. E. Murphy-Ullrich, J. Cell Biol. 122, 923 (1993); O'Conner-McCourt, L, M. Wakefield J. Biol. Chem. 262, 14090 (1987); and J. Massague Curr. Biol. 1, 117 (1991)). Thrombospondin has been shown to bind and activate latent TGF-.beta., but does not neutralize cytokine activity. In contrast, decorin has been shown to neutralize the anti-proliferative activity of TGF-.beta. (Yamaguchi et al., Nature 346:281, 1990). Decorin has also been found to antagonize the action of TGF-.beta. in vivo using an experimental glomerulonephritis model (Border et al., Nature 360:361, 1992).
Bovine fetuin, is one of the first glycoproteins to be purified from fetal calf serum (FCS). Fetuin is analogous to human .alpha.-2 HS-glycoprotein (.alpha.2-HS) which is believed to be produced by the liver (Kellerman et al. J. Biol. Chem. 264:14121, 1989). Fetuin has been shown to promote cell growth in vitro without any known cell surface receptor (B. T. Puck, C. A. Waldren, C. Jones, Proc. Natl. Acad. Sci. U.S.A. 59, 192 (1968), and it has been shown to be a negative acute phase reactant (J. P. Lebreton, F. Joisel, J. P. Raoult, B. Lannuzel, J. P. Rogez, et al, J. Clin. Invest. 64, 118 (1979). M. Daveau, C. Davrinche, N. Djelassi, J. Lemetayer, N. Julen, et al, FEBS Lett. 273, 79 (1990)). Serum fetuin accumulates in bone (I. R. Dickson, A. R. Poole, A. Veis, Nature 256, 430 (1975), J. T. Triffitt, U. Gebauer, B. A. Ashton, M. E. Owen, Nature 262, 226 (1976), G. Rauth, O. Poschke, E. Fink, M. Eulitz, S. Tippmer, et al, Eur. J. Biochem. 204, 523 (1992)) with highest concentrations found during bone growth. It also enhances bone resorption in cultured bone explants in vitro (G. C. Colclasure, W. S. Lloyd, M. Lamkin, W. Gonnerman, R. F. Troxler, et al, J. Clin. Endocrin. Metabolism 66, 187 (1988), and increases adipogenesis (A. J. Cayatte, L. Kumbla, M. T. R. Subbiah, J. Biol. Chem 265, 5883 (1990)). Inflammation is associated with significantly reduced serum fetuin concentrations (J. P. Lebreton, F. Joisel, J. P. Raoult, B. Lannuzel, J. P. Rogez, et al, J. Clin. Invest. 64, 118 (1979). M. Daveau, C. Davrinche, N. Djelassi, J. Lemetayer, N. Julen, et al, FEBS Lett. 273, 79 (1990)). Serum fetuin concentrations are also depressed in patients with Paget's disease, an affliction of increased bone turnover which leads to disordered and thickened bone (B. A. Ashton, R. Smith, Clin. Sci. 58, 435 (1980)). In a subset of osteogenesis imperfecta patients, the loss of bone is associated with elevated serum fetuin levels.
The cDNA sequence encoding the bovine fetal protein fetuin has been reported (K. M. Dziegielewska et al., J. Biol. Chem. 265:4254, 1990). The deduced amino acid sequence indicates that the protein is a single chain preceded by a signal sequence. The sequence of fetuin shows over 70% similarity to human .alpha..sub.2 HS glycoprotein.
Decorin is a small chondroitin-dermatan sulphate proteoglycan consisting of a core protein and a single glycosaminoglycan chain. The expression of high levels of decorin in Chinese hamster ovary cells has a dramatic effect on their morphology and growth properties and this effect has been attributed in part to the ability of decorin to bind transforming growth factor-.beta. (Y. Yamaguchi et al., Nature 346:281, 1990). The administration of decorin has been found to inhibit the increased production of extracellular matrix and attenuate manifestations of glomerulonephritis in an experimental glomerulonephritis model (W. A. Border, Nature 360:361, 1992).